Fetal Alz-50 Clone 1 Interacts with the Human Orthologue of the Kelch-like Ech-Associated Protein

Abstract

The fetal Alz-50 reactive clone 1 (FAC1) protein exhibits altered expression and subcellular localization during neuronal development and neurodegenerative diseases such as Alzheimer's disease. Using the yeast two-hybrid screen, the human orthologue of Keap1 (hKeap1) was identified as a FAC1 interacting protein. Keap1 is an important regulator of the oxidative stress response pathway through its interaction with the Nrf family of transcription factors. An interaction between full-length FAC1 and hKeap1 proteins has been demonstrated, and the FAC1 binding domain of hKeap1 has been identified as the Kelch repeats. In addition, FAC1 colocalizes with endogenous Keap1 within the cytoplasm of PT67 cells. Exogenously introduced eGFP:hKeap1 fusion protein redistributed FAC1 to colocalize with eGFP: hKeap1 in perinuclear, spherical structures. The interaction between FAC1 and hKeap1 is reduced by competition with the Nrf2 protein. However, competition by Nrf2 for hKeap1 is reduced by diethylmaleate (DEM), a known disrupter of the Nrf2:Keap1 interaction. DEM does not affect the ability of FAC1 to bind hKeap1 in our assay. These results suggest that hKeap1 regulates FAC1 in addition to its known role in control of Nrf2. Furthermore, the observed competition between FAC1 and Nrf2 for binding hKeap1 indicates that the interplay between these three proteins has important implications for neuronal response to oxidative stress.Fetal ALZ-50 Reactive Clone 1 (FAC1) 1 is a transcriptional regulator with enhanced expression patterns in both developing and degenerating neurons (1-4). FAC1 transcript and protein levels have been shown to be much higher in the developing fetal brain compared to the adult brain (5). Similarly, FAC1 is elevated in response to nerve growth factor induced differentiation of PC12 cells (6). FAC1 protein expression is enhanced during several neurodegenerative diseases. For instance, FAC1 protein is elevated in affected brain regions of patients with Alzheimer's disease (AD) (4). Furthermore, β-amyloid treatment of † This project was supported by NIH Grants NS41202, AG13208, and NS10572 to K.J.-S. and NS42902 to R.B.© 2004 American Chemical Society * Author to whom correspondence should be addressed [telephone (215) 898-4196; fax (215) 573-2050; jordan@path.dental.upenn.edu]. ⊥ These authors contributed equally to this work. ‡ Department of Pathology, University of Pennsylvania § Department of Oral Medicine, University of Pennsylvania ‖ University of Pittsburgh Medical Center 1 Abbreviations: AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; ARE, antioxidant response element; BTB, broad complex, tramtrack, and bric á brac; DEM, diethylmaleate; DGR, double-glycine repeats; FAC1, fetal Alz-50 reactive clone 1; GST, glutathione-S-transferase gene; GFP, green fluorescent protein; IVT, in vitro translated; KLD, Kelch-like domain; Keap1, Kelch-like Ech-associated protein; hKeap1, human Keap1; Phd, phalloidin; PBS, phosphatebuffered saline; POZ, pox virus genes that have zinc fingers; TBS, T...

1H, 15N and 13C backbone resonance assignments of the Kelch domain of mouse Keap1

“…In addition to Nrf2, several novel targets of the Kelch domain of Keap1 have been identified recently. These include FAC1 (Strachan et al 2004), PGAM5 , WTX (Major et al 2007;Camp et al 2012), prothymosin α (Padmanabhan et al 2008), p62 (Komatsu et al 2010) and PALB2 (Ma et al 2012). The assignments reported here will also serve as important starting points for studying the interaction of Keap1 with these proteins.…”

Stress-Activated Cap'n'collar Transcription Factors in Aging and Human Disease

“…Similarly, because Keap1 targets Nrf2 for degradation through a physical association with Cullin3, disrupting the Keap1-Cullin3 interface should also lead to Nrf2 stabilization and target gene induction. However, because Keap1 has other binding partners and ubiquitination substrates in addition to Nrf2 (62, 72, 296, 297), inhibiting Keap1 dimerization or Cullin3 binding may also affect the protein stability or activity of those proteins, with unpredictable functional consequences. Thus, disrupting the Keap1-Nrf2 interaction is likely the most specific intervention.…”

Binding of disordered proteins to a protein hub

“…In addition to NRF2, at least 10 other proteins have been shown to interact with the Kelch domain of Keap1 to date. These include WTX17, p6218, PGAM519, PALB220, FAC121, PTMA22, IKKβ23 and BCL224. Several of these partners have been identified to disrupt the low affinity site 2-Kelch domain interaction, allowing NRF2 to promote cytoprotective gene expression.…”